EIAV P26 deletion vaccine and diagnostic

ABSTRACT

Disclosed herein is a vaccine which provides immunity to mammals from infection and/or disease caused by a lentivirus, such as equine infectious anemia virus, human immunodeficiency virus (HIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) or simian immunodeficiency virus (SIV) said composition comprising a deletion in a gene that blocks replication of the virus in vivo. Said composition allows differentiation between vaccinated and non-vaccinated, but exposed, mammals and provides safety and immunity when administered as a vaccine to mammals. Preferably said composition encompasses at least one deletion in a lentivirus which allows mammals to be safely vaccinated and provides protection from exposure to wild-type lentiviruses. It also encompasses a marker vaccine in which a foreign gene is inserted into the gene-deleted region, said inserted gene providing a diagnostic tool for use in vaccinated mammals and, potentially, protection from infection from a foreign disease. The scope of the invention encompases an EIAV vaccine that allows equines to be safely vaccinated and protected from disease without converting to a seropositive status on the Coggin&#39;s Test or any other test which measures p26, said p26 antigen being expressed in disease-producing wild-type EIAVs. Additionally, said EIA vaccine virus cannot cause clinical disease in mammals or spread or shed to other mammals including equines. Finally, this invention encompasses a marker vaccine in which vaccinated equines can be distinguished from non-vaccinated equines by detection of a foreign gene in the vaccinated animals. A diagnostic test to detect this foreign gene or gene product is also described.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention pertains to a vaccine composition which providesimmunity from clinical disease signs and/or infections caused bylentiviruses including but not limited to Equine Infectious Anemia Virus(EIAV), Human Immunodeficiency Virus (HIV), Feline ImmunodeficiencyVirus (FIV), Bovine Immunodeficiency (BIV) and Simian ImmunodeficiencyVirus (SIV) or any other similar lentivirus. More specifically, butwithout limitation hereto, the invention relates to an Equine InfectiousAnemia Virus (EIAV) composition which provides immunity from clinicaldisease signs and/or infection with EIAV, and which composition allowsdiagnostic differentiation between vaccinated and non-vaccinated butexposed or diseased mammals, and which allows the vaccinated animal totest negative using a Coggins test or other similar test that detectsp26-specific antibodies

[0003] 2. Brief Description of the Prior Art

[0004] Lentiviruses are a subfamily of retroviruses that causepersistent infection and chronic disease in numerous types of mammalsincluding humans (HIV), equines (EIA), felines (FIV), bovines (BIV) andmonkeys (SlV). All of the diseases are spread by blood transmission.EIAV causes persistent infection and chronic disease in horses worldwide. With EIAV, the blood transmission occurs by biting flies and otherinsects carrying virus particles from one horse to another. The firstcycle of disease (clinical episode) in an infected horse usually occurswithin 42 days after exposure to the virus. This first cycle is usuallyreferred to as the acute stage of EIA and is characterized by pyrexia,thrombocytopenia, anorexia, depression and high plasma viremia levels.Anemia is not usually detected at this stage. Resolution of this firstfebrile episode is normally observed after 1 to 5 days and occursconcomitantly with a dramatic drop in the amount of plasma-associatedvirus. Following the acute stage, some animals may remain clinicallynormal while others go on to experience multiple bouts of illness inwhich severe anemia may accompany pyrexia, thrombocytopenia, edema, anddramatic weight loss, and death. In instances of persistent infection bya lentivirus, as illustrated by EIAV, nucleotide sequence data hasrevealed a high mutation rate of the lentivirus genome as reported byPayne et al, Virology, 1987: 161, p. 321-331 which is incorporatedherein by reference. With EIAV infections, it is generally thought thatneutralizing antibodies aid in the selection of new antigenic virusvariants during persistent infections. Also, with EIAV infections,serologically distinct variants of EIAV emerge possibly through immuneselection pressure operating on random viral genome mutations. Withoutbeing bound to any particular theory, it is believed that horses thatshow no further clinical signs of disease have developed a mature immuneresponse that can protect against the virus and its known mutations.

[0005] As a member of the lentivirus subfamily of retroviruses, EIAV isuseful as a model for the pathogenicity, immunology, vaccinology,treatment and prevention of HIV. The disease is significant in its ownright because horses that demonstrate exposure to EIAV as measured bytesting for anitbodies in the blood (Coggins Test or similar p26detecting test) are either required to be destroyed or strictlyquarantined. As a result of the Coggins Test requirement and its broaduse throughout the world, especially in testing performance horses thatare transferred into and out of the United States, it is critical thatany effective EIA vaccine not be able to seroconvert horses to apositive Coggins Test or to any other test that detects p26. Therefore,for vaccines useful in protecting against EIA, it is important to eitherdelete all or part of the gene expressing p26 or block its expression bydeleting regulator genes or inserting stop codons or foreign genes. Itis expected that use of the methods described herein can providevaccines for the other lentiviruses (HIV, FIV, BIV and SIV) that canelicit immune responses that are effective and that can be distinguishedfrom viral infections.

[0006] As with other lentiviruses such as HIV, BIV, FIV and SIV, thegenetic organization of EIAV classifies it as a complex retrovirus. TheEIAV genome contains the canonical gag, pol, and env genes common to allretroviruses, and three accessory genes (S1, S2 and S3). The gag geneencodes the core proteins of the virus designated as Matrix Antigen(MA), Capsid Antigen (CA), Nucleocapsid (NC) and a protein designatedp9. The env gene encodes the viral envelope proteins (gp90 and gp45).The pol gene encodes the enzymes that replicate the viral genome,designated as Deoxy UTPase (DU), Reverse Transcriptase (RT) andIntegrase (IN). The S1 open reading frame (ORF) encodes the viral Tatprotein, a transcription trans activator that acts on the virallong-terminal-repeat (LTR) promoter element to stimulate expression ofall viral genes. The S3 ORF encodes the Rev protein, apost-transcriptional activator that acts by interacting with its targetRNA sequence, named the Rev-responsive element (RRE), to regulate viralstructural gene expression. The S2 gene is located in the pol-envintergenic region immediately following the second exon of Tat andoverlapping the amino terminus of the Env protein (see FIG. 1). Itencodes a 65 amino acid protein with a calculated molecular mass of 7.2kDa. S2 appears to be synthesized in the late phase of the viralreplication cycle by ribosomal leaky scanning of a tricistronic mRNAencoding Tat, S2 protein, and Env protein, respectively.

[0007] The gag-encoded Capsid Antigen (CA) or p26 protein comprises thecapsid shell of the virion that is enclosed in the viral envelope andthat contains the viral RNA genome. Homologous CA proteins are presentin HIV, FIV, BIV and SIV and are also encoded by the respective gaggenes. As noted above, detection of antibodies to the p26 antigen is thebasis for the Coggins Test and certain other commercial tests used todiagnose EIA in horses. To be compatible with current regulatoryguidelines, it is critical that any EIAV vaccine not stimulateseroconversion in these diagnostic assays based on detection of serumantibodies to EIAV p26. The p26 antigen is highly antigenic in thatextremely small amounts of its presence in a vaccine can stimulateantibody responses and seroconversion in diagnostic assays. Attempts toextract or delete p26 antigen from a pool of EIAV have not beenpractical for vaccine production. Therefore, it would seem that onecould eliminate it by deletion of the gag gene, a segment of the gaggene that interferes with the expression of p26 or deletion orinactivation of a control gene that regulates the expression of p26.However, it has been determined by the inventors that deletion of thegag gene or segments thereof produces an EIAV particle that is unable toreplicate in vitro (tissue culture) or in vivo. Therefore, simplydeleting or blocking expression of p26 makes growth of EIAV for vaccineproduction impractical if not impossible.

[0008] To provide protection from disease and protection from infection,envelope proteins (Env) are considered the proteins of choice, as theseproteins are the predominant immune targets during infection. Byprotection from disease is meant that a mammal exposed to the virus doesnot demonstrate clinical signs (fever, lethargy, anemia, death, etc.),but does carry virus particles in its blood, which particles aredetectable by a reverse transcriptase polymerase chain reaction test(RT-PCR). By protection from infection is meant that a mammal exposed tothe virus does not demonstrate clinical signs of EIA and does notcontain RT-PCR-detectable virus particles in blood. The major envelopeproteins of EIAV are gp90 and gp45. These are proposed as the protectiveantigens or protective components of EIAV. By the term protectivecomponents is meant antigens from that produce either protection fromdisease or protection from infection as indicated above. It is thereforeimportant that any effective lentivirus vaccine contain amounts of thelentiviral Env proteins (such as gp 120, gp90 or gp45) effective toprotect mammals from disease caused by the lentivirus. The protectivecomponents from EIAV include but are not limited to gp90 and gp45. TheCapsid Antigen (p26) is not a protective component of EIAV and, becauseof its ability to stimulate a significant antibody response, the vaccineof the present invention preferably lacks the ability to stimulate p26antibodies in an equid.

[0009] It would seem obvious to prepare a vaccine by purifying out theEnv proteins, especially gp90 and gp45 for EIAV. Indeed, vaccinescomprising preparation from which gp90 and gp45 have been purified outof the EIAV have been attempted with extremely limited success. Issel etal (J. Virol. June 1992, p 3398-3408) reports that a gp90/gp45 vaccineprotected ponies from infection caused by homologous EIAV (the subunitswere derived from the same EIAV strain as was used for challenge).However, these subunit-containing vaccines did not protect horses fromeither disease or infection when challenged with a heterologous EIAVstrain. In fact, the latter produced enhanced disease signs. Theenhancement of disease by the subunit EIAV vaccine corroborates findingswith SIV and FIV subunit vaccines that appear to enhance disease postchallenge. Issel et al (ibid) concludes that perfecting a subunitvaccine for lentiviruses (e.g., HIV, FIV, EIA, BIV and SIV) poses asignificant challenge because of the subunit enhancement effect.

[0010] Issel, et al (ibid) also reports the prevention of infection by awhole-virus EIAV vaccine. However, vaccination of horses with thisvaccine produces horses that are Coggins Test positive (p26 positive).As mentioned previously, due to the eradication program in effect in theU.S., horses testing positive for p26 are either euthanized or strictlyquarantined. Additionally, the amount of virus included in said vaccinewas 1 milligram, an amount not commercially feasible. Therefore, thiswhole-virus vaccine is not compatible with regulatory requirements orcommercialization.

[0011] A donkey virus vaccine has been in use by the Chinese for morethan 20 years. This vaccine was developed by using total EIAV geneticmaterial from donkey leukocyte attenuated EIAV infected cells andribonucleic acid from virus in peripheral blood of donkey-adapted EIAVfrom infected donkeys (see Xinhua News Agency, May 6, 1999). As would beexpected, this vaccine produces a; p26 positive response (Coggin's Testpositive) in vaccinated horses or other vaccinated equids. Such avaccine is not acceptable in those countries where equids are tested byCoggins assays or other p26-specific antibody tests. In addition,numerous countries will not accept live vaccines for veterinaryapplications.

[0012] Since there has been no effective and safe method for immunizingmammals against disease or infection caused by lentiviruses,particularly equines against EIA, and since lentivirus diseases,especially HIV, FIV and EIA are such a wide-spread and significantdiseases world-wide, there remains a long-felt need to prepare such avaccine.

[0013] The vaccine of this invention provides a successful vaccinecomposition that effectively and safely immunizes mammals from diseasescaused by lentiviruses. The vaccine of the present invention protectsequines from EIA wherein vaccinated equines can be differentiated fromwild-type infected equines, which does not convert said equines toCoggins Test positive and which does not replicate in vivo. It is fullyenvisioned that the vaccines taught by the present invention can be usedfor production of any lentivirus vaccines, including vaccines for HIV,FIV, BIV and SIV.

DESCRIPTION OF THE FIGURES

[0014]FIG. 1 is a schematic representation of EIAV designated EIAV_(UK)

[0015]FIG. 2 is a circular map of infectious clone EIAV_(UK)

[0016]FIG. 3a is a linear schematic of the molecular clone EIAV_(UK)

[0017]FIG. 3b is a linear schematic of molecular clone EIAV_(UK) withthe CMV promoter.

[0018]FIG. 3c is a linear schematic of molecular clone pCMVEIAV_(UK)with the CA gene deleted.

[0019]FIG. 3d is a linear schematic of molecular clone pCMVEIAV_(UK)ΔCAwith the Amp Resistance gene replaced by the Kanamycin Resistance gene.

[0020]FIG. 3e is a linear schematic of the p26-deleted Proviral ClonepCMV.ΔCA.neo.

[0021]FIG. 4 is a circular map of the p26-deleted Proviral ClonepCMV.ΔCA.neo.

[0022]FIG. 5a is a linear schematic of the EIAV_(UK) molecular clone.

[0023]FIG. 5b is a linear schematic representation of the EIAV_(UK)clone with the CMV promoter insert (CMVEIAV_(UK)).

[0024]FIG. 5c is a linear schematic representation of the pCMVEIAV_(UK).vis2.

[0025]FIG. 5d is a linear schematic representation of the Proviral Clonecontaining the Kanamycin Resistance Marker.

[0026]FIG. 5e is a linear schematic representation of the finalpCMVEIAV_(UK).Vis2.neo Proviral Construct.

[0027]FIG. 6 is a Circular map of the final pCMVEIAV_(UK).Vis2.neoProviral Construct.

[0028]FIG. 7 is the nucleotide and amino acid map of the CA gene/EIAVp26.

[0029]FIG. 8 is the nucleotide and amino acid map of the CA gene/Visnap30.

[0030]FIG. 9 is a comparison of the homology between p26 of EIAV and p30of Visna virus.

[0031]FIG. 10a is a Western Blot of p26-deleted clones, Visna chimericclones & subcdones of EIAV using gp90 & p26 monoclonal antibodies as thedetector.

[0032]FIG. 10b is a Western Blot of several p26-deleted clones, Visnachimeric clones & subclones of EIAV using p30 monoclonal antibody as thedetector.

[0033]FIG. 11 is a graph demonstrating the Reverse TranscriptaseActivity of various subcdones of ED cells transfected withpCMVEIAVUK.Vis2neo Proviral Construct.

SUMMARY OF THE INVENTION

[0034] This invention encompasses a safe and effective vaccine thatproduces immunity to mammals from infection and/or disease caused by alentivirus. Examples of the lentivirus can be equine infectious anemiavirus, human immunodeficiency virus (HIV), feline immunodeficiency virus(FIV), bovine immunodeficiency virus (BIV) or simian immunodeficiencyvirus (SIV) said vaccine comprising a deletion in the gene encoding theCapsid Antigen. More specifically, the invention encompasses a vaccinecomprising a deletion that produces a lack of ability of the lentivirusto express the Capsid Antigen and to replicate in vivo while retainingthe lentivirus protective components. Also, the vaccine allowsdifferentiation between vaccinated and non-vaccinated, but exposed,mammals and provides safety and immunity when administered as a vaccineto mammals. By the term safe is meant that vaccination with of mammalswith vaccines of the present invention does not produce infection,disease or any other adverse reaction in the vaccinated mammals. Saidvaccine encompasses at least one deletion in a lentivirus, which allowsmammals to be safely vaccinated and provides protection from exposure towild-type lentiviruses. The invention further encompasses a lentiviruswith a deletion in the gag gene, specifically a deletion that results inan inability of the lentivirus to express the Capsid Antigen (CAprotein) in vivo or in vitro. Finally, the EIAV vaccine of the presentinvention lacks the ability to stimulate p26 antibodies in an equid.

[0035] In a preferred embodiment, the invention encompasses a vaccinefor effectively and safely immunizing mammals from EIA, said compositioncomprising a gene-deleted EIAV construct wherein said gene-deletedconstruct interrupts virus replication in vivo and blocks the expressionof p26 in vivo while retaining the EIAV protective components. As such,vaccinated equines would be protected from disease caused by EIAV andnot convert to a seropositive status on the Coggins Test or any othertest that measures p26 antibodies. As used herein, the term EIA refersto the disease Equine Infectious Anemia and the term EIAV refers to theEquine Infectious Anemia Virus that causes the disease. Additionally,said EIAV vaccine cannot cause clinical disease in mammals or spread orshed to other mammals including equines.

[0036] A more specific embodiment of the invention is a vaccine whereinthe lack of ability to express p26 antigen results from one or more genedeletions within the gag gene, one or more deletions within a genehaving a regulatory effect on gag CA production, an insertion of one ormore stop codons into the gag CA gene or a gene regulating CAproduction, or insertion of a foreign gene into the gag CA gene or agene regulating CA production. By insertion of a foreign gene is meantthat the gene being inserted is not a gene associated with EIAV. Saidforeign gene is obtained from a non-EIAV organism.

[0037] Additionally, it is expected that further deletions could be madesuch that the EIAV in the vaccine contained multiple deletions includingbut not limited to a deletion in the gag gene affecting the expressionof p26.

[0038] Finally, it is expected that said gene deletions (deletedregions) could serve as potential points for insertion of foreign genesto produce a multiple-protective vaccine. This means that a singlevaccination with the EIAV vaccine carrying a foreign gene (e.g.,influenza hemagglutinin (HA) gene) could protect the mammal from boththe lentivirus disease (e.g., HIV or EIA) and the disease associatedwith the foreign gene insert (e.g., human or equine influenza).

DETAILED DESCRIPTION OF THE INVENTION

[0039] This invention provides a vaccine for effectively and safelyimmunizing mammals against diseases caused by lentiviruses selected fromthe group consisting of EIAV, HIV, FIV, BIV and SIV, said vaccinecomprising a gene-deleted lentivirus construct. The inventionencompasses a vaccine comprising a deletion that produces a lack ofability of the lentivirus to replicate in vivo and retains thelentivirus protective components. By lentivirus protective components ismeant the protective antigens associated with the envelope of thelentiviruses including but not limited to gp120, gp90 and gp45. Theinvention encompasses a lentivirus that is unable to express the CapsidAntigen (CA protein) in vivo.

[0040] A deletion can be produced in the lentivirus genome by usingspecific restriction endonucleases to remove all or part of one or moregenes. A preferred gene for removal is the gene encoding the CapsidAntigen (CA). Such gene deletion can be accomplished by using PCR,ligation and PCR cloning; to delete the selected gene sequence.Restriction endonucleases can also be used to remove specific portionsof genes once the gene sequence of the lentivirus and the gene sequenceof the gene to be excised are known. Using specific restrictionendonucleases, the gag gene can be removed in whole or part.Additionally, a stop codon can be inserted into the gene, preferably atthe 5′ end wherein the stop codon causes the gene not to express its CAprotein. Additionally, a foreign gene from another lentivirus or anunrelated virus can be inserted into the gene-deleted region producing amultiply protective vaccine. In the latter case, the invention describesthe deletion of a region of the EIAV genome large enough to insert agene expressing a protective antigen from a non-EIAV organism,preferably a virus. Therefore, the HA gene from equine influenza A2 orA1 can be inserted into the gag CA region allowing expression of gp90and gp45 of EIAV as well as HA of A1 and A2 equine influenza. This willprovide a vaccine that can protect from disease in equines caused byEIAV and equine influenza viruses. Also, genes from equine herpesviruses types 1, 2, and 4 can be inserted into the EIAV construct toprovide protection against disease of equines caused by EIAV and equineherpes viruses. Other equine viruses which could have genes encoding forprotective antigens inserted in the EIAV include but are not limited toequine arteritis, encephalomyelitis viruses (Eastern, Western,Venezuelan and Rift Valley Fever virus). Genes encoding protectiveantigens from parasites (Sarcocystis neurona that causes EquineProtozoal Encephalitis or EPM, Neospora heugesi that is also possiblyrelated to EPM, Toxoplasma gondii, etc.) can also be inserted into anEIAV construct to protect against these diseases. Finally, genesencoding for bacterial diseases of horses, including but not limited toStreptococcus equi and Clostridium tetani, can be inserted into an EIAVconstruct to provide multiple disease protection. It is expected thateven a gene encoding for an immunostimulatory protein (immunomodulatorgene) or glycoprotein can be inserted into the gene-deleted region inorder to enhance the immunity provided by the virus construct.

[0041] Broadly described, a method for deleting a gene of a lentivirus(eg the CA gene) and insertion of a foreign gene utilizes the techniquesof PCR, ligation, and a method of PCR cloning.

[0042] Primers are designed to amplify a region of a promoter-lentivirusgenome upstream of the CA open reading frame (ORF). Additional primersare used to amplify the region of the promoter-lentivirus genomedownstream of the CA. The amplified PCR products are purified usingagarose gel electrophoresis and ligated together. A final round of PCRis performed using the 5′ primer of the upstream fragment, and the 3′primer of the downstream fragment, followed by gel purification. Thefinal product would comprise a representative size of the gag gene witha deletion of the CA open reading frame. The PCR product is gel purifiedand digested with specified restriction endonucleases such that it canbe ligated with a plasmid that had been digested with the samerestriction enzymes or enzymes producing the same blunt ends. Theligated insert is preferably added to a lentivirus clone comprising apromoter and genes allowing for selection of clones (e.g., antibioticresistance genes) thus producing a promotor-lentivirus clone. Then thepromoter-lentivirus clone is transformed into competent bacterial cellsand colonies of the bacteria are screened for insertion of the genes.Clones may be genetically sequenced to verify that the CA region hadbeen deleted and an insert had been made.

[0043] A gene-deleted construct could be commercially produced (producedin large scale) by transfecting susceptible tissue culture cells,harvesting the fluids and formulating the fluids with an adjuvant.Optionally, the harvest fluids may be inactivated with art-knowninactivating agents such as formalin, binary ethyleneimine,beta-propiolactone, thimerasol and psoralen. By gene-deleted constructis meant a lentivirus in which a gene is non-functional due to adeletion, an insertion of a stop codon, or production of a geneinsertion in which the deleted gene is replaced by a gene from anothervirus.

[0044] If said gene-deleted lentivirus cannot replicate in vitro, tissueculture cells may be transfected with the construct using transfectingagents such as DEAE dextran, GenePORTER™ (Gene Therapy Systems), etc.,to incorporate the necessary genomic material into the cell DNA suchthat the cells produce lentivirus antigens. For transfection, tissueculture cells are seeded into wells of tissue culture vessels (egplates), exposed to the gene-deleted construct or thegene-deleted/gene-inserted construct in the presence of a transfectingagent, incubated to allow transfection and then overlayed with aselection medium. Selection media is defined as any nutrient medium thatcontains components to kill non-transfected cells but does not inhibitgrowth of transfected cells. To accomplish this, generally, gene-deletedconstructs contain inserts of a resistance gene in order to allow theconstruct to grow in said selection media. Selection media can containantibiotics, antimicrobials and selective antibiotics. Once transfectedcells have been selected and are replicating they are tested forproduction of protective antigens as well as for the absence ofexpression of the deleted gene product. Those clones demonstrating thesecharacteristics are then expanded in selection media by removing thecells from their initial container, diluting them and replanting theminto larger containers. For instance, initial transfection may becarried out in 24 well tissue culture plates. After selection of clones,the surviving transfected cells are passaged to 6 well plates, 25 cm²flasks, 75 cm² flasks and then to roller bottles (1700 cm² or larger).Transfected cells should consistently produce the virus construct,indicating a stable transfected or producer cell. After the transfectedcell clones have been demonstrated to be stable, stable-transfectedMaster Cells (also referred to as persistently infected cells by variousregulatory agencies) can be prepared for expansion into Working Cellsand Production Cells. Working Cells are defined as those cells that areused to prepare Production Cells. Production Cells are the cells used tomanufacture vaccines. Master Cells, Working Cells and Production Cellsare all generally stored in liquid nitrogen for retaining viability andstability of the transfecting clone.

[0045] In the practice of this invention, a vaccine comprising agene-deleted construct lacks the ability to replicate in vivo and,possibly, in vitro. As should be realized by the foregoing, this type ofdeletion, if producing an inability to replicate or grow in vitro,requires transfection and cloning as described above.

[0046] The following is an illustrative but non-limiting description ofa lentivirus that is unable to express the Capsid Antigen protein (CA orp26) in vivo. It has been determined that with EIAV, a deletion in theCA such that the p26 is not expressed results in a gene-deletedconstruct that cannot replicate in vitro or in vivo. For this reason, itis expected that such a CA deleted lentivirus would have to be producedin a stable transfected cell line. This means that it would have to betransfected as described above in order to produce the stabletransfected cell line.

[0047] This invention more specifically encompasses a vaccine whereinthe lack of ability to express p26 antigen is produced by one or moregene deletions within the gag gene or one or more deletions within agene having a regulatory effect on gag CA production, or an insertion ofone or more stop codons or insertion of a foreign gene.

[0048] It is expected that further deletions could be made such that theEIAV in the vaccine composition contained multiple deletions includingbut not limited to a deletion in the gag gene affecting the expressionof p26. Finally, it is expected that said gene deletions (deletedregions) could served as potential points for insertion of foreign genesto produce a multiply-protective vaccine and a very important featurefor EIAV, a marker vaccine. A marker vaccine is a vaccine that containsa foreign gene that produces antibody in the mammal receiving avaccination, said antibody being detected by a diagnostic test and beingused to distinguish a vaccinated equid from a non-vaccinated equid and avaccinated equid from an infected equid. With EIAV, it is preferred toinsert a CA gene from a different lentivirus that does not cross-reactwith p26 in the Coggins Test or equivalent tests. Therefore, insertionof the p30 gene from a different lentivirus such as a Visna virus wouldbe expected to allow an EIAV vaccine to be used for vaccination ofmammals, preferably equids. Said equids would demonstrate no p26antibody in the Coggins Test or any other test measuring the presence ofantibody to p26 antibodies, and would, additionally, demonstrateantibody to p30 which could be detected by an enzyme linkedimmunosorbant assay (ELISA), immunodiffusion test, fluorescent antibodytest (FA), or any other test that can be used to detect antibodies inmammals.

[0049] It is expected that the gag gene-deleted constructs discussedabove will not grow or replicate in vitro. Therefore, in order toproduce large quantities for manufacturing purposes, the clonedconstructs can either be expressed by bacterial cells or by mammaliancells (tissue culture). The process of transformation has been describedbriefly above and is described in detail in the EXAMPLES. Production ofa stable transfected tissue culture cell line (persistently infectedMaster Cell) is preferable and is accomplished by transfecting mammaliancells in tissue culture. A preferred technique for EIAV constructs isdescribed in the examples to follow.

[0050] The resulting p26 deleted construct can be employed in a vaccinefor effectively and safely immunizing equines from EIAV, said vaccinecomprising a gene-deleted EIAV construct wherein said gene deletionblocks the expression of p26 in vivo.

[0051] Vaccine viruses or virus constructs of this invention can befurther treated with inactivating agents such as formalin, betapropiolactone, binary ethyleneimine, thimerasol or any other thateffectively inactivates viruses. Such agents can be used in amountsvarying from 0.00001% to 0.5%, preferably from 0..00001% to 0.1% andmore preferably from 0.00001% to 0.01%.

[0052] Additionally, adjuvants or immunomodulators/immunostimulators maybe added to the vaccine to enhance the immune response produced by thevaccine. Adjuvants can be selected for the group consisting of polymerssuch as Carbopol®-based, HAVLOGEN® and POLYGEN®, block co-polymers,oil-in-water such as EMULSIGEN® or EMULSIGEN® PLUS, water-in-oil,aluminum salts, lipid-based, lipoprotein, endotoxin-based andcombinations thereof. Immunomodulators and imuno-stimulators include butare not limited to Corynebacteria pyogenes and extracts or subunitsthereof, parapox viruses and extracts or subunits thereof, modified liveviruses that stimulate interferon production, as well as cytokines.

[0053] The vaccines of this invention can be administered by any route.For instance, they can be administered intramuscularly, subcutaneously,intradermally, intranasally, orally, intravenously or intraperitoneally.It is preferable to administer the vaccines either intramuscularly,subcutaneously, orally or intranasally.

[0054] Other antigens may be added to the vaccines such that amulti-component vaccine can be produced. In order to accomplish this,antigens from other viruses, bacteria or parasites are formulated withadjuvants or other excipients and then combined with the EIAV constructof this invention. Therefore, this invention encompasses an EIAVconstruct combined with antigens from the group selected from equineinfluenza (A1 and A2), equine herpes virus (subtypes 1, 2, 3 or 4),equine arteritis virus, eastern equine encephalomyelitis, western equineencephalomyelitis, Venezuelan equine encephalitis, Rift Valley FeverVirus, Sarcocystis neurona, Neospora hugesi, Toxoplasma gondii, Giardialamblia, Streptococcus equi, Streptococcus zooepidemicus, Rhodococcusequi, Clostridium botulinum, Clostridium tetani, Clostridium difficileor any other equine disease-producing agent. The Clostridium botulinumcan include types A, B, C, D, E, and/or F.

[0055] Finally, it is within the scope of this invention that adiagnostic test can be used to differentiate vaccinated equines fromnon-vaccinated and/or infected equines by measuring the presence orabsence of antibodies to the deleted gene protein. Also, a PCR-baseddiagnostic test could be used to detect the presence or absence of thegenes or gene sequences in body fluids or tissues from the equine and,thus, detect whether an equine had been infected with EIAV or vaccinatedwith the composition of this invention. The diagnostics of choicemeasure the presence or absence of p26 antibodies in an equine.Additionally, if an inserted gene is from a non-equine organism such asa Visna virus, a protein product of the non equine organism could bemeasured. An example described herein includes the insertion of the p30gene from Visna virus wherein the p30 can be detected in vaccinatedequines but is not present in non-vaccinated or EIAV infected equines.

[0056] Diagnostic differentiation can be measured by developing animmunoassay, an antibody-detecting assay (e.g., indirect fluorescentantibody, immunodiffusion, agar diffusion, electrophoresis) or aPCR-based assay known to the art. An example of an immunoassay is anenzyme linked immunosorbant assay (ELISA) that detects and/orquantitates antibodies to specific proteins in serum, blood or tissues.ELISA technology could also be used to detect the presence or absence ofvirus-associated antigens in the blood, serum or tissues. Byvirus-associated antigens is meant the presence or absence of a geneexpression product such as the p26 protein of EIAV or p30 protein ofVisna virus or in the case of the p26 or p30 genes, respectively.PCR-based assays have been used to measure the presence or absence ofgenes or gene sequences in the blood, serum or tissues of an equine,thus indicating that a horse had been infected or vaccinated, as thecase may be. For this particular embodiment, an ELISA would detect thepresence of antibodies to the p26 or p30 proteins. If p26 antibodieswere present in horses that were tested it would indicate that the horsehad been infected with EIAV. Horses that had been vaccinated with agene-mutated EIAV construct containing a non-functional p26 gene wouldnot contain p26 antibodies in their serum. Horses that had beenvaccinated with a gene-mutated EIAV construct containing a p30 geneinsertion would contain p30 antibodies in their serum. Thus, vaccinatedhorses could be differentiated from infected horses. The PCR-basedassays would be used to detect the presence or absence of gene sequenceswithin the horse. For instance, if a horse had been infected with awild-type EIAV, it would contain the gene sequence for wild-type p26.However, equines immunized with vaccines comprising a gene-mutated EIAV,particularly one wherein the p26 gene comprised deletions or specificmutations would not contain the gene sequence for wild-type p26.Alternatively, horses that had been vaccinated with a gene deleted EIAVconstruct containing a p30 gene insertion would contain the p30 genesequence in their serum.

[0057] These and other aspects of the invention are further illustratedby the following non-limiting examples. In the examples and throughoutthe specification, parts are by weight unless otherwise indicated.

EXAMPLE 1

[0058] Construction of the p26 Deletion Mutant Proviral Clone designatedas pCMV.ΔCA.neo: In order to determine whether deletion of all or partof the CA gene was possible, it was decided to delete the entire p26gene from EIAV. The molecular clone EIAV_(UK) as described by Cook etal. Journal of Virology 72(2): 1383-1393, 1998 which is incorporatedherein by reference, was used for derivation of the proviral clone. FIG.2 displays a circular map of the EIAV_(UK) molecular clone. FIG. 3adisplays a linear schematic in order to demonstrate the methods used forthe constructs described in this example. FIG. 6 shows the specificsequence of the CA gene and the amino acid sequence of p26 of the EIAvirus that it encodes.

[0059] The procedure for the construction of the p26 deletion mutantproviral clone (pCMV.ΔCA.neo) was as follows. First, the CMV promoterwas inserted into the 5′ LTR region through a process of PCR, ligation,and PCR cloning. Primers CMV3′Blunt (SEQ ID No. 1) and 5′CMVBssH (SEQ IDNo. 2) were used to amplify the CMV promoter from the plasmid pRC/CMV(InVitrogen). PCR conditions were set up as follows in thin-walled 0.5ml PCR tubes (PGC Scientific): 40.6 μl dH₂O, 5 μl cloned Pfu DNAPolymerase 10× reaction buffer, 0.8 μl 25 mM Deoxy-A,C,G,T (nucleotide)tri-phosphate (dNTP) mixture, 2.5 μl each primer (100 ng/μl), 1 μltemplate DNA (10 ng/μl) 2.0 μl cloned Pfu DNA Polymerase (2.5U/μl-Stratagene). Amplification was performed in a Hybaid thermocyclerand consisted of 30 cycles of: 94° C.-20seconds, 60° C.-20 seconds, 72°C.-1 minute. Primers LTRBlunt5′ (SEQ ID No. 3) and MA3′Tth (SEQ ID No.4) were used to amplify a region of the EIAV_(UK) clone encompassing theportion of the genome including the final 31 base pairs of the terminalredundancy region (R region) through the MA open reading frame insimilar reaction conditions. The two PCR products (50 μl) were gelpurified on a 0.8% agarose gel with GeneClean (Bio101). The two purifiedPCR products were set up in individual kinase reactions as follows: 5 μlDNA, 2 μl ATP, 2 μl 10× Protein Kinase buffer (New England Biolabs), 10μl dH₂O, and 1 μl Protein Kinase. The reaction product was incubated a37° C. 2 hours. The resulting kinased products were purified throughchloroform extraction and ethanol precipitated. The resultant products(3 μl) were ligated together overnight (16° C.) at their individualblunt ends with T4 ligase (New England Biolabs) in the followingreaction mixture: 1 μl 10× T4 ligase buffer, 2 μl dH₂O, and 1 μl T4ligase. A second round of PCR using the primers CMV5′BssH (SEQ ID 2) andMA3′Tth (SEQ ID 4) amplified the final product to be cloned into theEIAV_(UK) clone. The reaction conditions were as stated above using 1 μlof the ligation reaction. This final PCR product (50 μl) was gelpurified again on a 0.8% agarose gel. The purified PCR product wasdigested with the restriction enzymes BssHll and Tth111l in thefollowing manner: 17 μl PCR product, 2 μl BssHll 10× buffer(NEB), and 2μl BssHll (NEB), incubated at 50° C. for 2 hours, chloroform extractedand ethanol precipitated. The digestion was completed as follows: 16 μlDNA (BssHll digested), 2 μl 10× reaction buffer #4 (NEB), 2 μl Tth111l,incubated at 65° C. for 3 hours. The EIAV_(UK) clone (500 ng) waspartially digested with Mlul (New England Biolabs). This was conductedthrough incubation at 37° C. for 5 minutes in the following reactionmixture: 1 μl 10× # reaction buffer, 1 μl of restriction enzyme, 2 μl ofdH₂O and immediate submersion on ice followed by gel purification. Theappropriate size band was then completely digested with Tth111l in areaction mixture consisting of 1 μl 10× # 4 reaction buffer (NEB), 1 μlof restriction enzyme and 2 μl of dH₂O. The resulting fragment was gelpurified on a 0.8% agarose gel. The promoter fragment (3 μl) was ligatedinto the EIAV_(UK) clone (3 μl) with T4 ligase in a mixture of 1 μl 10×T4 ligase buffer, 2 μl dH₂O, and 1 μl T4 ligase. The resulting ligationproduct (4 μl) was transformed into competent DH5α bacterial cells (100μl). The transformation procedure consisted of: incubation on ice for 30minutes, heat shock at 42° C. for 45 seconds, incubation on ice for 2minutes, addition of 900 μl SOC borth (a media supplement containing 20%bacto-tryptone, 5% bacto-yeast, 0.5% NaCl, 2.5 mM KCl, 10 mM magnesiumchloride and 20 mM glucose), incubation at 37° C. for 1 hour, and 200 μlplated on LBAmp plates. Clones were sequenced to verify correct promoterarrangement as schematically represented in FIG. 3b

[0060] The PCR, ligation, PCR method of cloning was used to delete theCapsid Antigen (CA) sequence. Primers gag441 (SEQ ID No. 5) and MAT (SEQID No. 6) were used to amplify a 398 bp region of themolecularly-modified EIAV designated as CMVEIAVUKgenome upstream of theCA open reading frame. PCR conditions were set up as follows in PGCScientific thin-walled 0.5 ml PCR tubes: 40.6 μl dH₂O, 5 μl cloned PfuDNA Polymerase 10× reaction buffer, 0.8 μl 25 mM dNTP mixture, 2.5 μleach primer (100 ng/μl), 1 μl template DNA (10 ng/μl) 2.0Kl cloned PfuDNA Polymerase (2.5 U/μl-Stratagene). Amplification was performed in aHybaid thermocycler. Primers p9f5′ (SEQ ID No. 7) and p9f3′ (SEQ ID No.8) were used to amplify a 357 bp region of the CMVEIAV_(UK) genomedownstream of the CA encoding region in a similar reaction mixture.These two PCR products (50 μl) were gel purified on a 0.8% agarose gelwith GeneClean (Bio 101). The two purified PCR products (3 μl) wereligated together overnight (16° C.) with T4 ligase (New England Biolabs)in the following reaction mixture: 1 μl 10× T4 ligase buffer, 2 μl dH₂O,and 1 μl T4 ligase. A final round of PCR was performed using the gag441primer (SEQ ID 5) and p9f3′ primer (SEQ ID 8). The ligated sequence,when in the correct orientation would yield a PCR product ofapproximately 755 bp. This deletes the CA open reading frame from basepairs 846-1550 (EIAV base pair correlation, not plasmid). The PCRproduct was gel purified on a 0.8% agarose gel with GeneClean. Thepurified fragment was digested with Tth111l and BsrGl in the followingmanner: 15 μl PCR product, 2 μl BSA, 2 μl 10× buffer #2 (NEB), and 2 μlBsrGl (NEB), incubated at 37° C. for 3 hours, chloroform extracted andethanol precipitated. The digestion was completed as follows: 16 μlDNA(BsrGl digested), 2 μl 10× reaction buffer #4 (NEB), 2 μl Tth111l,incubated at 65° C. for 3 hours, and gel purified in the same mannerpreviously mentioned. The CMVEIAV_(UK) clone was digested with the samerestriction enzymes and gel purified in a similar format. The twofragments (3 μl each) were ligated together with T4 ligase in a mixtureof 1 μl 10× T4 ligase buffer, 2 μl dH₂O, and 1 μl T4 ligase, andtransformed into competent DH5α bacterial cells (100 μl). Thetransformation procedure consisted of: incubation on ice for 30 minutes,heat shock at 42° C. for 45 seconds, incubation on ice for 2 minutes,addition of 900 μl SOC broth, incubation at 37° C. for 1 hour, and 200μl plated on LBAmp plates. Individual clones were screened for insert.Clones were sequenced to verify that the CA region had indeed beendeleted as schematically diagrammed in FIG. 3c. The symbol Δ identifiesthe deletion.

[0061] The original proviral DNA carried an ampicillin resistance marker(Amp^(r)). Because this would not be the ideal marker for a vaccine usedin mammals, it was replaced with a Kanamycin resistant marker (Kan^(r))using the following procedure. The proviral DNA was subdoned into akanamycin-resistant vector designated as pLG339/SPORT (Cunningham et al.Gene, 124: 93-98, 1993). The vector was digested with the restrictionenzymes Mlul and EcoRl (New England Biolabs). The proviral clones werealso digested fully with EcoRl and partially digested with Mlul. Theplasmids (500 ng) were each partially digested individually throughincubation at 37° C. for 5 minutes in the following reaction mixture: 2μl 10× #1 reaction buffer, 1 μl of restriction enzyme (Mlul), 12 μl ofdH₂O and immediate submersion on ice followed by gel purification. Theappropriate size band was then completely digested with EcoRl in areaction mixture consisting of 1 μl 10× #2 reaction buffer, 1 μl ofrestriction enzyme and 2 μl of dH₂O. The desired fragments were gelpurified on a 0.8% agarose gel with GeneClean. The proviral DNA (4 μl)and vector (2 μl) were ligated together overnight (16° C.) with T4ligase (New England Biolabs) in the following reaction mixture: 1 μl 10×T4 ligase buffer, 2 μl dH₂O, and 1 μl T4 ligase. The ligation product (4μl) was transformed into competent DH5α bacterial cells (100 μl). Thetransformation procedure consisted of: incubation on ice for 30 minutes,heat shock at 42° C. for 45 seconds, incubation on ice for 2 minutes,addition of 900 μl SOC broth, incubation at 37° C. for 1 hour, and 200μl plated on LBKan plates. Individual clones were screened for insertinto the proper Mlul site. FIG. 3d shows a schematic representation ofthis construct demonstrating the Amp resistance marker being replace bythe Kan resistance marker.

[0062] A neomycin resistance marker (Neo^(r)) was added in order toallow selection of clones in eukaryotic cells. The neomycin resistancemarker was excised from the commercial vector pRC/CMV (InVitrogen) usingthe restriction enzymes EcoRl and Xhol (New England Biolabs). The areaexcised from the pRC/CMV encompassed the entire neomycin open readingframe as well as the SV40 promoter, origin of replication, and SV40 polyA recognition sequence. The digestion was executed at 37° C. in areaction mixture which consisted of 500 ng pRC/CMV plasmid DNA, 2 μl 10×#2 reaction buffer, 2 μl BSA, 2 μl dH₂O, and 1 μl each of therestriction enzymes. The resulting kanamycin-resistant proviral clonewas digested with the restriction enzymes EcoRl and Sall (GIBCO BRL).Sall digested ends can ligate into Xhol digested ends. The digestion wascarried out in the following reaction mixture: 1 μl proviral DNA, 2 μl10× REACT 6 buffer, 2 μl BSA, 2 μl H₂O and 1 μl each restriction enzyme.The digested neomycin fragment and proviral clone were gel purified on a0.8% agarose gel with GeneClean, and ligated together at 16° C.overnight with T4 ligase in the following reaction mixture: 4 μlpurified proviral DNA, 3 μl purified neomycin insert DNA, 1.5 μl 10× T4ligase buffer, 5.5 μl dH₂O and 1 μl T4 ligase. The ligated DNA (6 μl)was transformed into competent DH5α bacterial cells (100 μl). Thetransformation procedure consisted of: incubation on ice for 30 minutes,heat shock at 42° C. for 45 seconds, incubation on ice for 2 minutes,addition of 900 μl SOC broth, incubation at 37° C. for 1 hour, and 200μl plated on LBKan plates. Individual clones were screened for insert. Aschematic representation of the p26 deleted Proviral Glone pCMV.ΔCA.neois shown in FIG. 3e with a circular map shown in FIG. 4.

EXAMPLE 2

[0063] Construction of an EIAV wherein a gene from a non-EIAVorganism isinserted into the deleted p26 region (designated as pCMV.Vis2.neo): Inorder to substitute a foreign gene into the Capsid Antigen region (CA)of the gag gene and perhaps, to produce a replicating Proviral Clonewith a p26 deletion, it was decided to insert the p30 gene from a Visnavirus, a lentivirus (non-EIAV organism) which does not produce apositive response on the Coggins Test. If the p30 could be adapted toreplace the mechanism for p26 of the EIAV, then a replicating proviralclone could be produced.

[0064] As in EXAMPLE 1, the backbone for the construction of theProviral Clone with the p30 of Visna inserted into the deleted p26region was EIAV_(UK) (Cook et al., ibid). A schematic diagram of thisstarting construct is shown in FIG. 5a.

[0065] The procedure for preparation of this EIAV construct was asfollows: The CMV promoter was inserted into the 5′ LTR region ofEIAV_(UK) through a process of PCR, ligation, PCR cloning as referencedpreviously. Primers CMV3′Blunt (SEQ ID No. 1) and 5′ CMVBssH (SEQ ID No.2) were used to amplify the CMV promoter from the plasmid pRC/CMV(InVitrogen). PCR conditions were set up as follows in PGC thin-walled0.5 ml PCR tubes: 40.6 μl dH₂O, 5 μl cloned Pfu DNA Polymerase 10×reaction buffer, 0.8 μl 25 mM dNTP mixture, 2.5 μl each primer (100ng/μl), 1 μl template DNA (10 ng/μl) 2.0 μl cloned Pfu DNA Polymerase(2.5 U/μl-Stratagene). Amplification was performed in a Hybaidthermocycler and consisted of 30 cycles of: 94° C.-20 seconds, 60° C.-20seconds, 72° C.-1 minute. Primers LTRBlunt5′ (SEQ ID No. 3) and MA3′Tth(SEQ ID NO. 4) were used to amplify a region of the EIAV_(UK) cloneencompassing the portion of the genome including partial R regionthrough the matrix open reading frame in similar reaction conditions.The PCR products (50 μl) were gel purified on a 0.8% agarose gel withGeneClean (Bio 101). The two purified PCR products were set up inindividual kinase reactions as follows: 5 μl DNA, 2 μl ATP, 2 μl 10×Protein Kinase buffer (New England Biolabs), 10 μl dH₂O, and 1 μlProtein Kinase. The reaction was incubated a 37° C. 2 hours. The kinasedproducts were purified through chloroform extraction and ethanolprecipitated. The resultant products (3 μl) were ligated togetherovernight (16° C.) at their individual blunt ends with T4 ligase (NewEngland Biolabs) in the following reaction mixture: 1 μl 10× T4 ligasebuffer, 2 μl dH₂O, and 1 μl T4 ligase. A second round of PCR using theprimers CMV5′BssH (SEQ ID No. 2) and MA3′Tth (SEQ ID No. 4) amplifiedthe final product to be cloned into the EIAV_(UK) clone. The reactionconditions were as stated above using 1 μl of the ligation reaction.

[0066] This final PCR product (50 μl) was gel purified again on a 0.8%agarose gel. The purified PCR product was digested with the restrictionenzymes BssHll and Tth111l in the following manner: 17 μl PCR product, 2μl BssHll 10× buffer(NEB), and 2 μl BssHll (NEB), incubated at 50° C.for 2 hours, chloroform extracted and ethanol precipitated. Thedigestion was completed as follows: 16 μl DNA (BssHll digested), 2 μl10× reaction buffer #4 (NEB), 2 μl Tth111l incubated at 65° C. for 3hours. The EIAV_(UK) clone (500 ng) was partially digested with Mlul(New England Biolabs). This was conducted through incubation at 37° C.for 5 minutes in the following reaction mixture: 1 μl 10× # reactionbuffer, 1 μl of restriction enzyme, 2 μl of dH₂O and immediatesubmersion on ice followed by gel purification. The appropriate sizeband was then completely digested with Tth111l in a reaction mixtureconsisting of 1 μl 10× # reaction buffer, 1 μl of restriction enzyme and2 μl of dH₂O. The fragment was gel purified on a 0.8% agarose gel. Thepromoter (3 μl) was ligated into the EIAV_(UK) clone (3 μl) with T4ligase in a mixture of 1 μl 10× T4 ligase buffer, 2 μl dH₂O, and 1 μl T4ligase. The ligation product (4 μl) was transformed into competent DH5αbacterial cells (100 μl). The transformation procedure consisted of:incubation on ice for 30 minutes, heat shock at 42° C. for 45 seconds,incubation on ice for 2 minutes, addition of 900 μl SOC broth,incubation at 37° C. for 1 hour, and 200 μl plated on LBAmp plates.Clones were sequenced to verify correct promoter arrangement. FIG. 5b isa schematic representation of the EIAV_(UK) clone with the CMV promoterinsert (CMVEIAV_(UK)).

[0067] The source of the Visna p30 capsid sequence was the pVisna clonepuc9-4.9V2 (Braun, M J et al, Journal of Virology, 61(12): 4046-4054,1987). The Visna p30 (7 μl containing 1 μg) was excised out of the cloneusing the restriction enzymes Apal and Tth111l in the followingreaction: 4 μl dH₂O, 1.5 μl BSA, 1.5 μl 10× #4 reaction buffer (NEB),0.5 μl Apal and Tth111l (NEB), incubated at 65° C. for 2 hou? 0.5 μlmore of Apal added to the reaction mixture and incubated at roomtemperature (25° C.) overnight. The desired fragment was gel purified ina 0.8% agarose gel with GeneClean. The CMVEIAV_(UK) clone (5 μlcontaining 1 μg) was digested with Blpl (NEB enzyme for Bpu11021l) andTth111l (NEB) in the following reaction mixture: 1.5 μl 10× buffer #4(NEB), and 1 μl BsrGl (NEB), 7.5 μl dH₂O, incubated at 37° C. for 3hours, chloroform extracted and ethanol precipitated. The digestion wascompleted as follows: 15 μl DNA(Blpl digested), 2 μl 10× reaction buffer#4 (NEB), 1 μl Tth111l 2 μl dH₂O, incubated at 65° C. for 3 hours. Thedigested proviral DNA was gel purified on a 0.8% agarose gel withGeneClean. The two fragments were ligated with T4 ligase in thefollowing mixture: DNA fragments (3 μl each) were ligated together withT4 ligase in a mixture of 1 μl 10× T4 ligase buffer, 2 μl dH₂O, and 1 μlT4 ligase. The ligation product (4 μl) was transformed into competentDH5α bacterial cells (100 μl). The transformation procedure consistedof: incubation on ice for 30 minutes, heat shock at 42° C. for 45seconds, incubation on ice for 2 minutes, addition of 900 μl SOC broth,incubation at 37° C. for 1 hour, and 200 μl plated on LBAmp plates.Individual clones were screened for insert and sequenced using dideoxysequencing and an ABI automatic sequencer to verify the entire visna p30open reading frame was inserted in the proviral clone correctly and inframe. FIG. 5c shows a schematic of the CMVEIAV_(UK).vis2.

[0068] The proviral DNA was subcloned into a kanamycin-resistant vectordesignated as pLG339/SPORT (Cunningham et al. Gene, 124: 93-98, 1993),incorporated herein by reference. The vector was digested partially withMlul and fully with EcoRl (New England Biolabs). The proviral cloneswere also digested fully with EcoRl and partially digested with Mlul.The plasmids (500 ng) were each partially digested individually throughincubation at 37° C. for 5 minutes in the following reaction mixture: 2μl 10× #2 reaction buffer, 1 μl of restriction enzyme, 12 μl of dH₂O andimmediate submersion on ice followed by gel purification. Theappropriate size band was then completely digested with EcoRl in areaction mixture consisting of 1 μl 10× #2 reaction buffer, 1 μl ofrestriction enzyme and 2 μl of dH₂O. The desired fragments were gelpurified on a 0.8% agarose gel with GeneClean. The proviral DNA (4 μl)and vector (2 l) were ligated together overnight (16° C.) with T4 ligase(New England Biolabs) in the following reaction mixture: 1 μl 10× T4ligase buffer, 2 μl dH₂O, and 1 μl T4 ligase. The ligation product (4μl) was transformed into competent DH5α bacterial cells (100 μl). Thetransformation procedure consisted of: incubation on ice for 30 minutes,heat shock at 42° C. for 45 seconds, incubation on ice for 2 minutes,addition of 900 μl SOC broth, incubation at 37° C. for 1 hour, and 200μl plated on LBKan plates. Individual clones were screened for insertinto the proper Mlul site. FIG. 5d shows a schematic of the proviralclone containing the kanamycin resistance marker.

[0069] In order to make the EIAV proviral construct morecommercially-acceptable, the kanamycin resistance marker was replacedwith a neomycin resistance marker. The neomycin resistance marker wasexcised from the commercial vector pRC/CMV (InVitrogen) using therestriction enzymes EcoRl and Xhol. This encompassed the entire neomycinopen reading frame as well as the SV40 promoter (SEQ ID No. 9), originof replication (SEQ ID. No. 10), and SV40 poly A recognition sequence(SEQ ID. No. 11). The digestion was executed at 37° C. in a reactionmixture that consisted of 500 ng pRC/CMV plasmid DNA, 2 μl 10× #2reaction buffer, 2 μl BSA, 2 μl dH₂O, and 1 μl each of the restrictionenzymes. The new kanamycin-resistant proviral clone was digested withthe restriction enzymes EcoRl and Sall (GIBCO BRL). Sall digested endscan ligate into Xhol digested ends. The digestion was carried out in thefollowing reaction mixture: 1 μg proviral DNA, 2 μl 10× REACT 6 buffer,2 μl BSA, 2 μl H2O and 1 μl each restriction enzyme. The digestedneomycin fragment and proviral clone were gel purified on a 0.8% agarosegel with GeneClean, and ligated together at 16° C. overnight with T4ligase in the following reaction mixture: 4 μl purified provirai DNA, 3μl purified neomycin insert DNA, 1.5 μl 10× T4 ligase buffer, 5.5 μldH₂O and 1 μl T4 ligase. The ligated DNA (6 μl) was transformed intocompetent DH5α bacterial cells (100 μl). The transformation procedureconsisted of: incubation on ice for 30 minutes, heat shock at 42° C. for45 seconds, incubation on ice for 2 minutes, addition of 900 μl SOCbroth, incubation at 37° C. for 1 hour, and 200 μl plated on LBKanplates. Individual clones were screened for insert. FIG. 5e shows aschematic drawing of the final pCMVEIAV_(UK).Vis2.neo proviral construct(hereinafter designated pCMV.Vis2.neo) and FIG. 6 shows the finalcircular map of this construct.

[0070] The pCMV.Vis2.neo proviral construct was tested for its abilityto replicate in vitro by using the standard replication assay asdescribed in EXAMPLE 1. As with the Proviral Clone pCMV.ΔCA.neo, thispCMV.Vis2.neo proviral construct did not replicate in vitro and wouldnot be expected to replicate in vivo. It was therefore decided todevelop a transfected cell line (persistently-infected cell line).

EXAMPLE 3

[0071] Transfection & Selection of Cell Lines: Transfection of an EquineDermal Cell Line

[0072] The p26-deleted Proviral Clone pCMV.ΔCA.neo and proviralconstruct pCMV.Vis2.neo were used to evaluate their ability to transfectcells in a manner similar to the wild-type EIAV_(UK). The procedure usedwas as follows.

[0073] One microgram of proviral clone or proviral construct DNA wasused to transfect an Equine Dermal (ED) cell line (ATCC CRL 6288). TheED cell line was grown in 6 well tissue culture plates seeded withbetween 2 and 4×10⁵ ED cells per well in 2 mL of the complete growthMinimum Essential Media with Earies salts (EMEM) plus 10% fetal calfserum, 100 units/mL of penicillin, 100 μg/mL of streptomycin (Gibco BRL15140-122) and 2 mm L-glutamine (Gibco BRL 25030-081). The plates wereincubated at 37° C. in a CO₂ incubator approximately 16 to 24 hoursuntil the cells are between 50 and 80% confluent. For each transfection,1 μg of DNA was diluted into 100 μL of OPTI-MEM I Reduced Serum Medium(Gibco BRL 18324-012) and 10 μL of Lipofectamine reagent (Gibco BRL18324-012) was added to 100 μL of OPTI-MEM I Reduced Serum Medium(OPTI-MEM RSM). The two solutions were mixed gently and incubated atroom temperature for 30 minutes to allow the DNA-liposome complexes toform. During this time, the ED cell cultures were rinsed once with 2 mLof OPTIMEM I RSM (GIBCO-BRL). For each transfection, 0.8 mL of OPTI-MEMI RSM was added to the tube containing the DNA-liposome complexes, thetube was mixed gently and the contents were overlayed onto the rinsedcells. No antibiotics were added during transfection. TheDNA-liposome/tissue cultures were incubated for 5 hours at 37° C. in aCO₂ incubator. Following incubation, 1 mL of complete growth MEMcontaining twice the normal concentration of serum was added to the cellculture without removing the transfection mixture. Twenty four hoursfollowing the start of transfection the medium was replaced with freshcomplete growth medium (EMEM). Starting at 48 to 72 hours posttransfection, aliquots of the tissue culture supernatants were taken atperiodic intervals and analyzed by using a standard reversetranscriptase (RT) assay as a measure of virus production. Supernatantsresulting in RT activity were titrated in an infectivity assay based oncell-ELISA readings as described by Lichtenstein et al, 1995. Neitherthe Proviral Clone pCMV.ΔCA.neo nor the proviral construct pCMV.Vis2.neoreplicated in tissue culture. The RT levels were less than or equal tothose of the negative control in tissue culture cells normally capableof being infected with EIAV, that were exposed to the culture mediumfrom the transfected cells. Therefore, it was determined that thedeletion of p26 produced a defective virus particle, unable to replicatein vitro or in vivo. In order to obtain particles for large-scalevaccine production, it was decided to produce a persistently-infectedcell line with the Proviral Clone pCMV.ACA.neo and proviral constructpCMV.Vis2.neo.

[0074] Transfection of COS cells

[0075] Virus particles were produced using Proviral Clone pCMV.ΔCA.neoand the proviral construct pCMV.Vis2.neo transfected in the monkey cellline COS-1 (ATCC_CRL 1650). Cells were plated at approximately 50%confluency into 60 mm plates (Falcon) 24 hours prior to transfection.Approximately 1 μg of proviral clone DNA (pCMV.ΔCA.neo or pCMV.Vis2.neo) was transfected into the cells using DEAE Dextran methodology.Briefly, a 50 mg/ml solution of DEAE dextran was diluted 1:50 (1 mg/mlfinal concentration) in Tris-buffered saline (TBS) with DNA and added tothe cells in serum-free media (DMEM). The DNA solution was incubated onthe cells for 1 hour at 37° C. in the presence of 5% CO₂ with rockingevery 15 minutes. Regular growth medium was replaced at this point.Forty-eight hours post-transfection the supernatants were assayed for RTactivity. The RT activity was detected in cell-free supernatant samplesusing the micro reverse transcriptase assay (Lichtenstein et al., ibid).Protein content was detected using a Western Blot Anlaysis procedure.For this procedure, virus particles were pelleted from 10 mls ofcell-free supernatant over a 20% glycerol cushion in an ultracentrifuge(Beckman SW41Ti rotor) at 50,000× g for 45 min. Pellets were lysed in100 l of lysis solution containing 10 mM NaCl, 1% Deoxycholic acid(DOC), 0.1% Sodium Dodecyl Sulfate (SDS), 25 mM Tris-HCl and 1%TritonX-100 and transferred to 1.5 ml eppendorf tubes. After lysis, thesamples were boiled in 20 μl of 6× SDS gel loading buffer and loadedonto a 12% SDS-polyacrylamide gel. Gradient purified EIAV_(PV) (1 μg)was also loaded onto the gel to serve as a marker for viral proteins.Electrophoresis was carried out at approximately 10 mA overnight withcooling. Proteins were transferred onto Millipore membranes usingBioRad's protein transfer cell system in a buffer containing 25 mM Tris,192 mM glycine, 20% methanol and 0.05% SDS. Transfer was completed after3 hours at 400 mA with cooling. EIAV proteins were detected usingmonoclonal antibodies. Prior to antibody incubation the blot was blockedin 5% blotto (5% drymilk, 5% FBS and 0.25% Tween-20 in 1× PBS) for 1hour at room temperature. Mouse monoclonal α-gp90 and α-p26 were usedtogether in 5% blotto for 1 hour at room temperature. Secondary antibodyα-mouse 1 gG conjugated with horse-radish-peroxidase (Sigma lot #115H8995) was incubated at room temperature for one hour. The blot waswashed for 3-5 minute periods in 1×PBS/0.025% Tween-20 between primaryand secondary antibody incubations. A one minute incubation at roomtemperature of the chemi-illuminescent substrate SuperSignal (Pierce lot#AE40027) followed the final wash after the secondary antibodyincubation. Exposure of the blot to film demonstrated that both gp90 andp26 were detectable in the EIAV_(PV) positive control; but only gp90 wasdetectable in the proviral clone pCMV.ΔCA.neo and the proviral constructpCMV.Vis2.neo. Production of the virus particles was observed throughboth RT activity and by Western Blot analysis.

[0076] Stable Transfections in CHO, C-33A & ED-MCS Cell Lines

[0077] Stable production of virus particles was attempted in three celllines; a human cell line C-33A (ATCC HTB-31), a chinese hamster ovarycell, CHO (ATCC CRL-9618), and an equine cell line ED-MCS .Transfections were all done in duplicate. Cells were consistentlymaintained in an incubator at 37° C. with 5% CO₂. Cell lines were seededonto 10 mm plates manufactured by Sarstedt and Falcon 24 hours prior totransfection at the following densities: CHO & C-33A 1×10⁶ cells/plate,ED-MCS 3.5×10⁵ cells/plate. Proviral clones, pCMV.Vis2.neo andpCMV.ΔCA.neo (20 μg/plate) were transfected into the cells using 55 μlof the reagent GenePORTER™ (Gene Therapy Systems) in serum-free DMEM(Gibco). Manufacturers' instructions were followed. Twenty-four hourspost-transfection media was changed from transfection media to selectionmedia (DMEM) which contained 800 μg/ml G-418 (Geneticin, Gibco BRL) and10% FBS (Hyclone). A plate that was not transfected was carried as acontrol for selection in the same media. Once the control plate had noviable cells present and the selected plates displayed colony formation,cells were passed into T75 flasks (Falcon) as bulk cultures. The levelof G-418 in the ED-MCS cells was increased to 1000 μg/ml due to rapidgrowth. Supernatants were analyzed throughout the selection period forRT activity and at individual points assayed for protein content throughWestern blot analysis. RT activity initially indicated highestproduction in the human and mouse cell lines. The equine dermal cellline proved to develop the most stable construct during long-termproduction, producing continuously the highest levels out topost-selection day 150. This experiment proved that tissue culture cellscan be transfected by the p26-deleted clone as well as by the chimerawherein a foreign gene from a Visna virus (p30) was inserted into thep26 region. Reverse trascriptase activity from these trasnfected cellsreached levels as high as 10,000 CPM/10 μl of tissue culture fluid. Thisis equivalent to RT activity produced by wild-type EIAV when transfectedinto tissue culture. Western Blot analysis was conducted as describedpreviously except that a second western blot was done in the same formatas before, re-probing the membrane with goat α-Visna p30 to detect theVisna chimera proteins. Secondary antibody was α-goat lgG wholemolecule-HRP (Sigma lot# 117H4831). The Visna p30 protein was detectedin the Visna chimeric proviral construct pCMV.Vis2.neo (See FIG. 10b).

[0078] Western Blot Analysis

[0079] Virus particles were pelleted from 10 mls of cell-freesupernatant over a 20% glycerol cushion in the ultracentrifuge SW41Tirotor (Beckman). Pellets were lysed in 100 μl of lysis solutioncontaining 10 mM sodium chloride (NaCl), 1% DOC, 0.1% Sodium DodecylSulafte (SDS), 25 mM Tris-HCl and 1% TritonX-100 and transferred to 1.5ml eppendorf tubes. After lysis, the samples were boiled in 20 μl of 6×SDS buffer gel loading buffer and loaded onto a 12% SDS-polyacrylamidegel. One microgram of gradient purified pony virus EIAV_(PV) was alsoloaded onto the gel to serve as a marker for viral proteins.Electrophoresis was carried out at approximately 10 mA overnight withcooling. Proteins were transferred onto Millipore membranes usingBioRad's protein transfer cell system in a buffer containing 25 mM Tris,192 mM glycine, 20% methanol and 0.05% SDS. Transfer was completed after3 hours at 400 mA with cooling. EIAV proteins were detected usingmonoclonal antibodies. Prior to antibody incubation the blot was blockedin 5% blotto (5% drymilk, 5% FBS and 0.25% Tween-20 in 1× PBS) for 1hour at room temperature. Mouse monoclonal α-gp90 and α-p26 were usedtogether in 5% blotto for 1 hour at room temperature. Secondary antibodyα-mouse lgG conjugated with horse-radish-peroxidase (Sigma lot #115H8995) was incubated at room temperature for one hour. The blot waswashed for 3-5 minute periods in 1XPBS/0.025% Tween-20 between primaryand secondary antibody incubations. A one minute incubation at roomtemperature of the chemi-illuminescent substrate SuperSignal (Pierce lot#AE40027) followed the final wash after the secondary antibodyincubation. Exposure of the blot to film demonstrated that both gp90 andp26 were detectable in the PV positive control; but only gp90 wasdetectable in the proviral clones (pCMV.Vis2.neo and pCMV.CA.neo), seeFIG. 10a The membranes were stripped through incubation in Glycine-Cl pH2.3 buffer (0.05M glycine 0.15M NaCl) for 45 minutes. The membranes werewashed in the same wash buffer for 7-5 minute periods and blocked in 5%blotto for 2 hours. The second western was done in the same format asbefore, re-probing the membrane with goat α-Visna p30 to detect theVisna chimera proteins. Secondary antibody was α-goat lgG wholemolecule-HRP (Sigma lot# 117H4831). The Visna p30 protein was detectedin the Visna chimeric proviral constructs (pCMV.Vis2.neo) see FIG. 10b.

[0080] The presence of gp90 indicates that these p26-deleted constructsproduce the protective antigen. Not only do they lack the ability toproduce p26 antibodies in animals but they also cause the animalsvaccinated with them to produce antibodies to p30. The presence of p30in an equine will indicate that the horse has been vaccinated. An assayto detect the presence of this p30 antibody can be developed in order todifferentiate horses that are vaccinated with the vaccines of thisinvention from horses that have not been vaccinated or horses that havebeen infected with wild-type EIAV. Additionally, a diagnostic thatdetects all or part of the p30 gene sequence or the p30 protein can beused similarly as a diagnostic tool.

EXAMPLE 4 Subcloning—Single Cell Cloning of the Stable Transfection

[0081] Stablely-selected Visna (pCMV.Vis2.neo) transfected ED-MCS cellswhich had been frozen back at day 40 of selection were thawed at 37° C.and seeded into a T75 flask in normal growth medium (no G-418). Cellswere grown at 37° C. with 5% CO₂ in G-418-negative medium for 48-hoursprior to plating for cloning. Cells were trypsonized from the T75flasks, counted, and plated onto 100 mm Falcon plates at densities ofapproximately 100 cells per plate. The cells were selected in mediumcontaining 800 μg/ml G-418. Media was changed approximately every fourdays and cells were grown in the plates until visible colonies hadformed. Independent colonies were trypsonized from the plates separatelythrough the use of cloning cylinders and seeded into separate cells ofFalcon 24-well plates. These were also selected in media containing 800μg/ml G-418. Approximately 7 days post-transfer the cell supernatantswere assayed for RT activity. The was conducted as follows:

[0082] For each 10 μl sample of cell-free supernatant to be assayed thefollowing is added: ³H-TTP (40 Ci/mmol)  1.5 μl dried in speedvac andvolume made up with the volume of water below 100 mM EGTA  5.0 μl 10XSalts  5.0 μl (2M Tris-Cl pH 8.0, 2M KCl, 1M MgCl₂, 1M DTT, 20% NP-40,DI Water) poly(rA).p(dT)₁₂₋₁₃  2.0 μl (5 units/ml ˜ .25 mg/ml) milliporewater 38.0 μl 50.0 μl

[0083] The mixture of supernatant (sample) and reaction mixture aremixed together and incubated at 37° C. for 1.5 hr-2.0 hr. The totalvolume is (˜60 μl) pipetted onto DEAE coated filter paper and allowed todry completely. The filters are then washed 3× for 15 minute each in 1×SSC and again allowed to dry completely. The filters are then immersedin scintillation fluid and the incorporated activity measured. As aresult of using this RT assay, the 12 “subclones” with the highest RTactivity were trypsonized and passaged into 6-well plates (Falcon),still selecting in 800 μg/ml G-418. Supernatants were analyzed for RTactivity after 4 days of selection in the 6-well plates. The 8 subcdoneswith the highest RT activity were trypsonized and passaged into T75flasks (Falcon) still selecting in 800 μg/ml G-418. Supernatants wereanalyzed for RT activity after 7 days of selection in the flasks. Theamount of G-418 was reduced at this passage point to 600 μg/ml.Selection was carried out for 4 more days, RT activity analyzed, and thelevel of G-418 lowered again to 400 μg/ml. After 7 days of selectionanother RT assay was performed on the 8 subcdones to monitor selection.Following 7 more days of selection, another RT assay was performed. The4 highest producing cell lines were passaged again, lowering the levelof G-418 to 200 μg/ml (the other 4 were frozen back). Thehighest-producing subclone, F-1V2.23, was producing a high level of RTactivity (between 4000 and 50,000 CPM per 10 μl of tissues culture fluidas shown in FIG. 11. This result indicates that the constructs of thisinvention can be produced in vitro in enough quantity to producecommercial vaccines.

[0084] The fact that the constructs of this invention were able todemonstrate the presence of the gp90 protective component and displayedsignificant EIAV RT activity provides assurance that a vaccine preparedaccording to this invention would be useful in protecting animals fromdisease and/or infection from lentiviruses, particularly EIAV.Additionally, it has been demonstrated that said vaccine lacks theability to stimulate antibodies to p26 and that it would produceantibodies to p30 so that vaccinated animals can be differentiated frominfected or non-exposed animals. Most importantly, the insertion of aforeign gene into the EIAV genome such that said foreign gene isexpressed indicates the usefulness of this lentivirus as a vector or asa virus construct into which multiple genes could be inserted. Such amultiple gene insertion could provide for an EIAV vaccine that protectsfrom multiple diseases.

[0085] Although the invention has been described in detail in theforegoing, for the purpose of illustration it is to be understood thatsuch detail is solely for that purpose and that variations can be madetherein by those skilled in the art without departing from the spiritand scope of the invention except as it may be limited by the claim.

1 11 1 24 DNA GIBCO 1 aatttcgata agccagttaa gcag 24 2 26 DNA GIBCO 2ctggcgcgcg atcgacgggc cagata 26 3 28 DNA GIBCO 3 ggcctttcta ataaatataattctctac 28 4 27 DNA GIBCO 4 aggcctctct tccttgtcct gacagcg 27 5 27 DNAGIBCO 5 tggccagaac acaggaggac aggtaag 27 6 29 DNA GIBCO 6 gatattcttcagagggctca gactgcttt 29 7 25 DNA GIBCO 7 cagactggtc ttgcgggccc attta 258 23 DNA GIBCO 8 catcctctac ttgatccttc tcc 23 9 226 DNA IN VITROGEN 9ccaggcaggc acaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa 60gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac 120catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc 180tccgccccat ggctgactaa ttttttttat ttatgcagag gccgag 226 10 86 DNA INVITROGEN 10 gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgagctattccaga 60 agtagtgacc aggctttttt ggaggc 86 11 131 DNA IN VITROGEN 11acttctttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa 60ataaagcatt tttttcactg cattctagtt ctggtttgtc caaactcatc aatgtatctt 120atcatgtctg t 131

What is claimed:
 1. A vaccine that produces protection from diseaseand/or infection caused by a lentivirus comprising a lentivirus thatlacks the ability to replicate in vivo.
 2. A vaccine that producesprotection from disease and/or infection caused by a lentiviruscomprising a lentivirus that lacks the ability to express a CapsidAntigen
 3. The vaccine according to claim 2 wherein the lack of abilityto express Capsid Antigen results from one or more deletions in a generegion selected from the group consisting of a portion of the gag gene,all of the gag gene and a gene regulating expression of the gag gene oran insertion of a stop codon in a gene affecting the expression of thegag gene.
 4. The vaccine according to claim 1 wherein the lentiviruslacks the ability to replicate in vitro.
 5. The vaccine according toclaim 2 wherein the lentivirus lacks the ability to replicate in vitro,6. The vaccine according to claim 4 wherein said gene-deleted lentivirusis produced in large quantities by a cell line transfected with thegene-deleted lentivirus.
 7. The vaccine according to claim 5 whereinsaid gene-deleted lentivirus is produced in large quantities by a cellline transfected with the gene-deleted lentivirus.
 8. The vaccineaccording to claim 1 which, safely and effectively immunizes mammalsagainst disease and/or infection caused by a lentivirus and furtherallows for differentiation between vaccinated, non-vaccinated andwild-type exposed mammals.
 9. The vaccine according to claim 2 which,safely and effectively immunizes mammals against disease and/orinfection caused by a lentivirus and further allows for differentiationbetween vaccinated, non-vaccinated and wild-type exposed mammals. 10.The vaccine according to claim 1 wherein said lentivirus is selectedfrom the group consisting of equine infectious anemia virus (EIAV),human immunodeficiency virus (HIV), feline immunodeficiency virus (FIV),bovine immunodeficiency virus (BIV) and simian immunodeficiency virus(SIV).
 11. The vaccine according to claim 2 wherein said lentivirus isselected from the group consisting of equine infectious anemia virus(EIAV), human immunodeficiency virus (HIV), feline immunodeficiencyvirus (FIV), bovine immunodeficiency virus (BIV) and simianimmunodeficiency virus (SIV).
 12. The vaccine according to claim 11wherein the lentivirus EIAV lacks the ability to express p.26 antigenand is safe and effective.
 13. The vaccine according to claim 12 whereinthe lack of the ability to express p26 antigen results from anon-functional gag gene.
 14. The vaccine according to claim 13 whereinthe non-functional gag gene results from a deletion in the gag gene. 15.The vaccine according to claim 13 wherein the deletion in the gag generesults from one or more deletions in a gene region selected from thegroup consisting of a portion of the gag gene, all of the gag gene and agene regulating expression of the gag gene or an insertion of a stopcodon in a gene affecting the expression of the gag gene.
 16. Thevaccine according to claim 11 wherein the gene-deleted EIAV furtherlacks the ability to replicate in vitro.
 17. A vaccine for effectivelyand safely immunizing equines from EIA, said vaccine comprising agene-deleted EIAV wherein said gene-deleted EIAV lacks the ability toexpress p26 and allows differentiation of vaccinated from wild typeexposed equines.
 18. The vaccine of claim 17 further comprising anadjuvant.
 19. The vaccine of claim 17 wherein the EJAV is inactivated.20. The vaccine of claim 17 comprising an inactivated EIAV and anadjuvant
 21. A method of immunizing mammals against disease produced byan EIAV comprising, administering to said mammals the vaccine of claim11.
 22. An EIAV vaccine that allows equines to be safely vaccinated andprotected from disease and/or infection without converting to aseropositive status on the Coggins Test or any other test which measuresp26 antibodies.
 23. A diagnostic to detect all or a portion of the gaggene of EIAV comprising a PCR probe for said gene.
 24. A diagnostic todifferentiate between a vaccinated and wild type exposed equinecomprising the PCR probe of claim
 23. 25. A method of preparing alentivirus vaccine comprising: 1) deleting all or a portion of a gaggene from the lentivirus; 2) transfecting a tissue culture with theresulting gene-deleted lentivirus to produce a persistently transfectedcell culture; 3) growing the persistently transfected cell culture; 4)harvesting the persistently-transfected cell culture; 5) optionallyinactivating the harvested cell culture; and optionally adjuvantiing theharvested cell culture.